CN109943604A - A method of improving glutamic acid fermentation conversion ratio and recovery rate - Google Patents
A method of improving glutamic acid fermentation conversion ratio and recovery rate Download PDFInfo
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Abstract
The invention belongs to glutamic acid production technical fields, disclose a kind of method for improving glutamic acid fermentation conversion ratio and recovery rate comprising following steps: step 1) prepares Corynebacterium glutamicum seed liquor, step 2 batch fermentation, and step 3) extracts glutamic acid.The method of the present invention improves glutamic acid fermentation conversion ratio and recovery rate.
Description
Technical field
The invention belongs to glutamic acid production technical fields, are related to a kind of side for improving glutamic acid fermentation conversion ratio and recovery rate
Method.
Background technique
Glutamic acid is a kind of acidic amino acid.Molecule includes two carboxyls, and chemical name is alpha-amido glutaric acid.Paddy ammonia
Acid is to find for inner Suo Xun 1856, is clear crystal, has delicate flavour, be slightly soluble in water, and is dissolved in hydrochloric acid solution, isoelectric point 3.22.
Largely it is present in grain protein, content is also more in animal brain.During the protein metabolism of glutamic acid in vivo
Critical role is accounted for, many important chemical reactions in animal, plant and microorganism are participated in.Sodium glutamate is commonly called as monosodium glutamate, is important
Tasty agents, to fragrance have humidification.Sodium glutamate is widely used in food flavor, not only can be used alone, but can and its
Its amino acid etc. is used in combination.For in food, there is flavouring effect.Concentration is 0.2%-0.5% in food, allows to take in for each person every day
Measuring (ADl) is micro- g kg of 0-120 (in terms of glutamic acid).General dosage is 0.2-1.5 gs/kg in food processing.
Corynebacterium glutamicum is the bacterial strain of glutamic acid fermentation, belongs to facultative aerobe, medium component and condition of culture are not
Together, product is also different, and glutamic acid fermentation condition optimizing is mainly two aspect of nutrient media components and Fermentation Process of Parameter control optimization.
During the fermentation, the fermentation character of strain itself is it some times happens that variation, causes batch indirect fermentation performance greatest differences occur.
Once strain fermentation characteristic changes, thallus will decline the adaptability and acid producing ability of environmental change, show as mending
Occurs the phenomenon that " only consume sugar, do not produce acid " after material, final aminoglutaric acid concentration is very low, causes fermenting property unstable.Glutamic acid
The early period of fermentation, main bacterial strain proliferation was rapid, and it is synthesis point that middle and later periods bacterial strain growth rate, which slows down, but glutamic acid synthesis is accelerated
Secrete the critical period of glutamic acid, that is, convert from accumulation type to acid type bacterial strain is produced.
It includes as follows to patented technology " a method of improve glutamic acid fermentation middle and later periods conversion ratio " before applicant
Step: inositol and dimethylformamide are added during the fermentation, and uses ultrasonication, passes through addition suitable two
Methylformamide is conducive to substrate molecule and is easier to enter cell connect with biological enzyme so that the permeability of cell membrane of thallus enhances
Touching, and then improve conversion ratio；Suitable inositol can strengthen the fixed reaction of CO2, promote the accumulation of glutamic acid, improve fermentation and turn
Rate.On the basis of the research, Fu Feng group continues to study fermentation conversion rate and recovery rate, it is intended to further excellent
In glutamic acid production technology.
Summary of the invention
In order to overcome the production acid efficiency of prior art bacterial strain further to be promoted, the invention proposes a kind of raising paddy
The method of propylhomoserin fermentation conversion rate and recovery rate.
The present invention is achieved by the following technical solution:
A method of improving glutamic acid fermentation conversion ratio and recovery rate comprising following steps:
Step 1) prepares Corynebacterium glutamicum seed liquor, step 2 batch fermentation, and step 3) extracts glutamic acid.
Further, the batch fermentation is film coupling dialysis batch fermentation.
Further, the step 2 batch fermentation, comprising:
By Corynebacterium glutamicum seed liquor with the inoculation access of 6-10% inoculum concentration equipped in the 30L fermentor of 21L fermentation medium
Fermented and cultured is carried out, it is 33 DEG C of fermentation temperature, ventilatory capacity 0.4vvm, speed of agitator 500r/min, fermentor and ceramic membrane is even
Connection adds the inositol of 1-2g/L and the magnesium carbonate of 0.5-1g/L when fermentation is to 12h, continues the 12h that ferments, by the hair in fermentor
Zymotic fluid is pumped out via centrifugal pump, then through ceramic membrane separation, obtains filtrate and concentration thallus, filtrate is drained into feed liquid storage tank,
Concentration thallus is returned fermentor, while adding fermentation tank culture medium into fermentor, continues the 12h that ferments, then add 1-2g/L
Inositol and 0.5-1g/L magnesium carbonate, continue ferment 12h, complete fermentation, obtain fermentation liquid, by fermentor fermentation liquid pass through
It is pumped out by centrifugal pump, then through ceramic membrane separation, obtains filtrate and concentration thallus, filtrate is drained into feed liquid storage tank.
Further, the group of the fermentation tank culture medium is divided into (mass percent): glucose 12%, corn pulp 3%, urea
0.5%, potassium dihydrogen phosphate 0.5%, ferrous sulfate heptahydrate 0.02%, manganese sulfate monohydrate 0.01%, fulvic acid 0.002%.
Further, the molecular cut off of the ceramic membrane is 10000-20000Da.
Further, the step 3) extracts glutamic acid, comprising: the liquid in feed liquid storage tank passes through ultrafiltration membrane ultrafiltration, receives
Collect filtered solution, is then condensed into the concentrate of original volume one third；Into the electric tank such as level-one, stream adds simultaneously plus concentrate is stated
Enter the concentrated sulfuric acid and adjust the pH 3.5 for making solution in equal electric tank, temperature control at 22 DEG C, by level-one isoelectric point tank liquid again according to
It is secondary to pass through second level isoelectric point tank, while concentrated sulfuric acid tune pH value is added, wherein second level isoelectric point tank pH control 3.3,10 DEG C of temperature；
Successively pass through three-level isoelectric point tank again by the liquid of second level isoelectric point tank, while concentrated sulfuric acid tune pH value is added, wherein three-level etc.
Electricity point tank pH control 3.2,5 DEG C of temperature；Centrifugation obtains the glutamic acid of crystallization, obtained by drying.
Preferably, the ultrafiltration retaining molecular weight is 300Da.
Preferably, the step 1) prepares Corynebacterium glutamicum seed liquor, includes the following:
Corynebacterium glutamicum is inoculated into seed culture medium, at 33 DEG C, 100rpm shaking table culture 12h, obtained glutamic acid rod
Bacillus seed liquor.
Preferably, the group of the Corynebacterium glutamicum seed culture medium is divided into (being below mass percent): glucose
6%, corn pulp 3%, potassium dihydrogen phosphate 0.2%, epsom salt 0.02%, manganese sulfate monohydrate 0.01%, 115 DEG C of sterilizings
15min。
The glutamic acid product of above method production is also claimed in the present invention.
Compared with prior art, the beneficial effect that the present invention obtains mainly includes but is not limited to the following aspects:
The present invention is used using film coupling dialysis batch fermentation, reduces the production of the substances such as by-product acetic acid, lactic acid in fermentation process
It is raw, fermentation efficiency is improved, and amino acid zymotic fluid transparency is high, impurity is few, and separation and Extraction is more simple, and the later period is easier
Obtain the product of purity is high.
Containing groups such as a large amount of phenolic hydroxyl groups, carbonyls in fulvic acid, electrolysis degree is higher, can promote glutamic acid synthesis process
It is middle to utilize O2As hydrogen acceptor, and then pyruvic acid is reduced as hydrogen acceptor, add suitable fulvic acid in fermentation medium, it can
The production quantity of by-product lactic acid and acetic acid is reduced, and then improves the yield of glutamic acid；
Inositol can strengthen the fixed reaction of CO2, weaken glyoxalic acid circulation, guarantee that tricarboxylic acid cycle is not disrupted and in a steady stream not
Cut-off largely accumulates glutamic acid by reduction of amination to α-ketoglutaric acid, improves fermentation conversion rate；
Magnesium carbonate adds the time as ferment middle, and cell enters secretion by accumulation type and produces acid type, and magnesium carbonate can be with by-product cream
Acid, acetic acidreaction decompose and generate carbon dioxide, can provide carbon dioxide, are in CO2 content and both maintain thallus eupnea
Effect also ensures that more CO2 are fixed and generates oxaloacetic acid and acetyl-CoA synthesizing citric acid, and it is required to provide synthesis glutamic acid institute
C4 dicarboxylic acids, improve fermentation conversion rate；The byproduct reactions such as magnesium carbonate and acetic acid, lactic acid, reduce the murder by poisoning to bacterial strain, mention
High fermentation efficiency；Magnesium ion is the activator of a variety of enzymes during bacterial strain synthesis glutamic acid, can stimulate strain growth and production
Acid；
The present invention adds inositol and magnesium carbonate in ferment middle, improves fermentation efficiency.
It is easy to operate using power technologies such as ultrafiltration membrane ultrafiltration concentration and concentrations in extraction process of the present invention, it is pure by separating
Chemical industry skill makes glutamic acid purity be 90% or more, and yield is 95% or more.
Detailed description of the invention
Fig. 1: the schematic diagram of fermentation process of the present invention；
Fig. 2: film is coupled influence of the dialysis fermentation to Fungal biodiversity；
Fig. 3: influence of each factor to saccharic acid conversion ratio；
Fig. 4: influence of each factor to glutamic acid yield.
Specific embodiment
In order to make those skilled in the art better understand the technical solutions in the application, having below in conjunction with the application
The technical solution of the application is clearly and completely described in body embodiment, it is clear that described embodiment is only this Shen
Please a part of the embodiment, instead of all the embodiments.Based on the embodiment in the application, those of ordinary skill in the art are not having
Every other embodiment obtained under the premise of creative work is made, should fall within the scope of the present invention.
Embodiment 1
A method of improving glutamic acid fermentation conversion ratio and recovery rate comprising following steps:
Corynebacterium glutamicum ATCC13761 is inoculated into seed culture medium, at 33 DEG C, 100rpm shaking table culture 12h is obtained
Corynebacterium glutamicum seed liquor；The group of the Corynebacterium glutamicum seed culture medium is divided into (being below mass percent): Portugal
Grape sugar 6%, corn pulp 3%, potassium dihydrogen phosphate 0.2%, epsom salt 0.02%, manganese sulfate monohydrate 0.01%, 115 DEG C of sterilizings
15min；
By Corynebacterium glutamicum seed liquor with 10% inoculum concentration inoculation access equipped with 21L fermentation medium 30L fermentor in into
Row fermented and cultured, fermentor and ceramic membrane is coupled by 33 DEG C of fermentation temperature, ventilatory capacity 0.4vvm, speed of agitator 500r/min,
When fermentation is to 12h, add the inositol of 2g/L and the magnesium carbonate of 1g/L, continue the 12h that ferments, by the fermentation liquid in fermentor via from
Heart pump pumps out, and then through ceramic membrane separation, obtains filtrate and concentration thallus, filtrate is drained into feed liquid storage tank, concentration thallus
Fermentor is returned, while adding fermentation tank culture medium into fermentor, is made and the fermentating liquid volume before being handled without centrifugal pump
It is identical, continue the 12h that ferments, then add the inositol of 2g/L and the magnesium carbonate of 1g/L, continue the 12h that ferments, completes fermentation, fermented
Liquid pumps out the fermentation liquid in fermentor via centrifugal pump, then through ceramic membrane separation, obtains filtrate and concentration thallus, will filter
Liquid is drained into feed liquid storage tank；In entire fermentation process, by stream plus GPE defoaming, while Feeding ammonia water controls the pH value of fermentation liquid
To 6.0, stream plus glucose solution control residual sugar amount are not less than 1%；The molecular cut off of ceramic membrane is 20000Da；
The group of the fermentation tank culture medium is divided into (mass percent): glucose 12%, corn pulp 3%, urea 0.5%, biphosphate
Potassium 0.5%, ferrous sulfate heptahydrate 0.02%, manganese sulfate monohydrate 0.01%, fulvic acid 0.002%；
Liquid in feed liquid storage tank collects filtered solution by ultrafiltration membrane ultrafiltration (ultrafiltration retaining molecular weight is 300Da), then dense
Shorten the concentrate of original volume one third into；It flows to add into the electric tank such as level-one and states concentrate, while concentrated sulfuric acid adjusting is added to make
PH Deng solution in electric tank is 3.5, and temperature is controlled at 22 DEG C, by the liquid of level-one isoelectric point tank again successively by electricity such as second levels
Point tank, while concentrated sulfuric acid tune pH value is added, wherein second level isoelectric point tank pH control 3.3,10 DEG C of temperature；By second level isoelectric point
The liquid of tank successively passes through three-level isoelectric point tank again, while concentrated sulfuric acid tune pH value is added, wherein three-level isoelectric point tank pH control
3.2,5 DEG C of temperature；Centrifugation obtains the glutamic acid of crystallization, obtained by drying.
Embodiment 2
A method of improving glutamic acid fermentation conversion ratio and recovery rate comprising following steps:
Corynebacterium glutamicum ATCC13761 is inoculated into seed culture medium, at 33 DEG C, 100rpm shaking table culture 12h is obtained
Corynebacterium glutamicum seed liquor；The group of the Corynebacterium glutamicum seed culture medium is divided into (being below mass percent): Portugal
Grape sugar 6%, corn pulp 3%, potassium dihydrogen phosphate 0.2%, epsom salt 0.02%, manganese sulfate monohydrate 0.01%, 115 DEG C of sterilizings
15min；
Corynebacterium glutamicum seed liquor is equipped in the 30L fermentor of 21L fermentation medium with the inoculation access of 8% inoculum concentration and is carried out
Fermented and cultured 33 DEG C of fermentation temperature, ventilatory capacity 0.4vvm, speed of agitator 500r/min, fermentor and ceramic membrane is coupled, hair
When ferment is to 12h, add the inositol of 1g/L and the magnesium carbonate of 0.5g/L, continue the 12h that ferments, by the fermentation liquid in fermentor via from
Heart pump pumps out, and then through ceramic membrane separation, obtains filtrate and concentration thallus, filtrate is drained into feed liquid storage tank, concentration thallus
Fermentor is returned, while adding fermentation tank culture medium into fermentor, is made and the fermentating liquid volume before being handled without centrifugal pump
It is identical, continue the 12h that ferments, then add the inositol of 1g/L and the magnesium carbonate of 0.5g/L, continue the 12h that ferments, completes fermentation, sent out
Zymotic fluid pumps out the fermentation liquid in fermentor via centrifugal pump, then through ceramic membrane separation, obtains filtrate and concentration thallus, will
Filtrate is drained into feed liquid storage tank；In entire fermentation process, by stream plus GPE defoaming, while Feeding ammonia water controls the pH of fermentation liquid
For value to 5.5, stream plus glucose solution control residual sugar amount are not less than 1%；The molecular cut off of ceramic membrane is 10000Da；
The group of the fermentation tank culture medium is divided into (mass percent): glucose 12%, corn pulp 3%, urea 0.5%, biphosphate
Potassium 0.5%, ferrous sulfate heptahydrate 0.02%, manganese sulfate monohydrate 0.01%, fulvic acid 0.002%；
Liquid in feed liquid storage tank collects filtered solution by ultrafiltration membrane ultrafiltration (ultrafiltration retaining molecular weight is 300Da), then dense
Shorten the concentrate of original volume one third into；It flows to add into the electric tank such as level-one and states concentrate, while concentrated sulfuric acid adjusting is added to make
PH Deng solution in electric tank is 3.5, and temperature is controlled at 22 DEG C, by the liquid of level-one isoelectric point tank again successively by electricity such as second levels
Point tank, while concentrated sulfuric acid tune pH value is added, wherein second level isoelectric point tank pH control 3.3,10 DEG C of temperature；By second level isoelectric point
The liquid of tank successively passes through three-level isoelectric point tank again, while concentrated sulfuric acid tune pH value is added, wherein three-level isoelectric point tank pH control
3.2,5 DEG C of temperature；Centrifugation obtains the glutamic acid of crystallization, obtained by drying.
Embodiment 3
Analysis method
Thallus assay: OD600nm。
Glutamic acid measurement: using the multi-functional glutamic acid of SBA-40C-glucose analyser measurement.
Residual glucose: it is measured with Fehling method.
PH value measurement: pH determination of electrode is used.
Control group uses normal fermentation mode, culture medium and parameter with experimental group (embodiment 1) unanimously, fermentation time 36h.
Film is coupled influence of the dialysis fermentation to Fungal biodiversity
In glutamic acid fermentation production process, there is also close relationships for biomass and production acid, and relatively high biomass can
Secrete more glutamic acid.As shown in Figure 2, before carrying out dialysis fermentation (starting to dialyse for 24 hours) for 24 hours in, normal fermentation and
The OD of dialysis fermentation600nmValue is continuously increased, and reaches maximum value (53 or so) to fermentation 18h.For control fermentation, after 18h
OD600nmStart slowly decline, for 24 hours after then rapid decrease, reason be mainly nutrients shortage and toxic metabolite
Inhibiting effect.Corresponding dialysis fermentation then completely on the contrary, with dialysis progress, the removal of harmful toxic matter and nutrients
Addition so that thallus starts to increase, the OD of thallus600nmValue starts to be continuously increased to 36h again reaches maximum (63.7), Zhi Houkai
Beginning is slowly drop down to fermentation ends.It can be seen that addition of the dialysis fermentation to the separation and nutriment that are metabolized harmful toxic matter etc.,
The growth vigor of thallus can be effectively improved, biomass is increased.By calculating the yield of glutamic acid, dialysis fermentation is
154.3g/L, and normal fermentation is 133.6g/L, improves 15.5 percentage points.
Embodiment 4
Influence of each factor to conversion ratio and glutamic acid yield:
Control group is set, in which:
Control, 1: inositol and magnesium carbonate are not added in fermentation process, remaining is the same as embodiment 1；
Control 2: magnesium carbonate is not added in fermentation process, remaining is the same as embodiment 1；
Control 3: fermentation medium does not add fulvic acid, remaining is the same as embodiment 1；
Experimental group is embodiment 1.Each group conversion ratio and aminoglutaric acid concentration are shown in Fig. 3-4, compared with compareing 1-3, the saccharic acid of experimental group
Conversion ratio and glutamic acid yield reach highest.Suitable fulvic acid is added in fermentation medium, can reduce by-product lactic acid
With the production quantity of acetic acid, and then improve glutamic acid yield；And inositol can strengthen the fixed reaction of CO2, it is mutual with magnesium carbonate
Saccharic acid conversion ratio can be improved in collaboration, reduces murder by poisoning of the by-product to bacterial strain, improves glutamic acid yield.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method for improving glutamic acid fermentation conversion ratio and recovery rate comprising following steps:
Step 1) prepares Corynebacterium glutamicum seed liquor, step 2 batch fermentation, and step 3) extracts glutamic acid.
2. the method according to claim 1, wherein the batch fermentation is film coupling dialysis batch fermentation.
3. method according to claim 1 or 2, which is characterized in that the step 2 batch fermentation, comprising:
By Corynebacterium glutamicum seed liquor with the inoculation access of 6-10% inoculum concentration equipped in the 30L fermentor of 21L fermentation medium
Fermented and cultured is carried out, it is 33 DEG C of fermentation temperature, ventilatory capacity 0.4vvm, speed of agitator 500r/min, fermentor and ceramic membrane is even
Connection adds the inositol of 1-2g/L and the magnesium carbonate of 0.5-1g/L when fermentation is to 12h, continues the 12h that ferments, by the hair in fermentor
Zymotic fluid is pumped out via centrifugal pump, then through ceramic membrane separation, obtains filtrate and concentration thallus, filtrate is drained into feed liquid storage tank,
Concentration thallus is returned fermentor, while adding fermentation medium into fermentor, continues the 12h that ferments, then add 1-2g/L's
The magnesium carbonate of inositol and 0.5-1g/L, continue ferment 12h, complete fermentation, obtain fermentation liquid, by the fermentation liquid in fermentor via
Centrifugal pump pumps out, and then through ceramic membrane separation, obtains filtrate and concentration thallus, filtrate is drained into feed liquid storage tank.
4. according to the method described in claim 3, it is characterized in that, the group of the fermentation medium is divided into (mass percent):
Glucose 12%, corn pulp 3%, urea 0.5%, potassium dihydrogen phosphate 0.5%, ferrous sulfate heptahydrate 0.02%, manganese sulfate monohydrate 0.01%,
Fulvic acid 0.002%.
5. according to the method described in claim 3, it is characterized in that, the molecular cut off of the ceramic membrane is 10000-
20000Da。
6. method according to claim 1 or 2, which is characterized in that the step 3) extracts glutamic acid, comprising: feed liquid storage
Liquid in tank passes through ultrafiltration membrane ultrafiltration, collects filtered solution, is then condensed into the concentrate of original volume one third；Toward level-one etc.
Stream is plus stating concentrate in electric tank, while the concentrated sulfuric acid is added and adjusts the pH 3.5 for making solution in equal electric tank, and temperature control is 22
DEG C, successively pass through second level isoelectric point tank again by the liquid of level-one isoelectric point tank, while concentrated sulfuric acid tune pH value is added, wherein two
Grade isoelectric point tank pH control 3.3,10 DEG C of temperature；Successively pass through three-level isoelectric point tank again by the liquid of second level isoelectric point tank, together
When be added concentrated sulfuric acid tune pH value, wherein three-level isoelectric point tank pH control 3.2,5 DEG C of temperature；Centrifugation obtains the glutamic acid of crystallization, dries
It does to obtain the final product.
7. according to the method described in claim 6, it is characterized in that, the ultrafiltration retaining molecular weight is 300Da.
8. method according to claim 1 or 2, which is characterized in that the step 1) prepares Corynebacterium glutamicum seed liquor,
Include the following:
Corynebacterium glutamicum is inoculated into seed culture medium, at 33 DEG C, 100rpm shaking table culture 12h, obtained glutamic acid rod
Bacillus seed liquor.
9. according to the method described in claim 8, it is characterized in that, the group of the Corynebacterium glutamicum seed culture medium is divided into
(being below mass percent): glucose 6%, corn pulp 3%, potassium dihydrogen phosphate 0.2%, epsom salt 0.02%, one
Water manganese sulfate 0.01%, 115 DEG C of sterilizing 15min.
10. the glutamic acid product produced according to method described in claim 1-9.
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